This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 09-234145, filed Aug. 29, 1997.
The present invention relates to an apparatus for detecting a target DNA, a target mRNA, etc. by using a DNA probe.
In recent years, a human genome project, i.e., an attempt to analyze the base sequence in all the human genes, is being carried out worldwide. The human genome analysis including the determination of the base sequence is a very complex and laborious work. It is said, however, that the human genome project will be completed at the beginning of the 21st century. Development of many novel technologies as well as improvement and automation of the analytical equipment greatly contributes to the promotion of the human genome project. A DNA chip technology is one of the newly developed analytical technologies.
The DNA chip is a chip prepared by spotting many kinds of DNA probes at predetermined positions on a substrate, e.g., a silicon wafer, by utilizing the lithographic technology used in the field of semiconductor devices. The DNA probe is formed of an oligonucleotide having a predetermined sequence of 4 kinds of bases constituting DNA, i.e., adenine (A), guanine (G), cytosine (C) and thymine (T). The sequence is complementary to the base sequence of the target DNA or mRNA. For example, if the sequence of AGCTT (5xe2x80x2xe2x86x923xe2x80x2) is used as a DNA probe, a DNA having a base sequence of AAGCT, which is complementary to the sequence of AGCTT noted above, is hybridized with the DNA probe so as to be selectively caught. Incidentally, the actual constituting unit of the DNA probe is nucleotide having the above-noted base portion (base portion+deoxyribose portion+phosphoric acid ester portion). For simplifying the description, however, nucleotide is represented by the base alone in the following description.
A method of immobilizing a DNA probe on the substrate is exemplified in, for example, xe2x80x9cScience 251:767-773xe2x80x9d published in February 1991. In this method, a DNA probe is formed on a flat substrate by utilizing a photochemical reaction. Let us describe briefly how to form a DNA probe having a 4-base length on a silicon substrate by this method with reference to FIGS. 1A to 1F.
In the first step, an amino group is formed on a silicon substrate S by treatment with silane, followed by allowing a photo-protective group X to be coupled with each amino group. Then, a desired position is selectively irradiated with an ultraviolet light by using a first mask M1. As a result, a protective group X at a desired position is removed so as to expose the amino group, as shown in FIG. 1A. Then, an optional DNA base (shown by K here) accompanied by the photolabile protecting group is reacted with the exposed amino group, as shown in FIG. 1B. As a result, a portion in which the base K accompanied by the photolabile protecting group X is coupled with the amino group and another portion in which the photolabile protecting group X alone is coupled with the amino group are formed on the substrate, as shown in FIG. 1C. Further, the portion in which the photolabile protecting group X alone is coupled with the amino group is selectively irradiated with light through a second mask M2 so as to selectively remove the photolabile protecting group X in the irradiated portion. Then, an optional DNA base (shown by L here) accompanied by the photolabile protecting group X is coupled with the exposed amino group, as shown in FIG. 1D. As a result, bases K and L accompanied by the photolabile protecting group X are immobilized to the substrate surface with an amino group (not shown) interposed therebetween, as shown in FIG. 1E. Further, similar operations are performed by using an optional DNA base (shown by M here) and another optional DNA base (shown by N here). As a result, a DNA probe having a length of two bases is immobilized to the substrate surface. Still further, similar operations are repeated to stack bases in a three dimensional direction so as to form DNA probes each having a length of four bases, said DNA probes having different base sequences depending on the process units, as shown in FIG. 1F. For forming a base sequence having a length of, for example, eight bases, photolithography using 32 masks and the photoreaction are repeated 32 times so as to form DNA probes having all the desired base sequences. It is theoretically possible to form efficiently DNA probes each having an optional base length and an optional base sequence on a single substrate by utilizing the above-described technology.
An improved method of manufacturing a DNA chip is disclosed in International Laid-open Application WO 93/096668 (Japanese translation version No. 7-506561). In this method, a DNA probe is immobilized on a silicon substrate having an amino group formed thereon by using a flow type channel block. The flow type channel block used in this method is a pattern plate having a plurality of slender channels. In the case of using the pattern plate, it is possible to immobilize the DNA probe along each of these channels. In this case, the base sequence of the DNA probe differs depending on the channel. However, the base sequence is the same over the entire length in a single channel. In this preferred mode of the conventional technology, an amino group is attached first to the substrate, followed by combining the substrate with a block having a plurality of channels arranged in parallel. Then, a process solution containing a base, which is a constituting unit of the selected DNA probe, is allowed to flow through a predetermined channel so as to immobilize the first base of the aimed DNA probe. Further, the substrate and the channel block are rotated relative to each other by a predetermined angle, e.g., 90xc2x0, followed by combining again the substrate and the channel block and subsequently immobilizing a base corresponding to the second base along the channel. A DNA probe having a desired base sequence can be prepared by successively repeating the above-noted steps. Also, a large number of DNA chips referred to previously can be manufactured in a single operation by combining the method described above with the photochemical reaction. It should also be noted that screening can be performed by using the DNA chip prepared by the above-noted method in combination with the channel block described above.
In order to prepare the conventional DNA chip, it is necessary to carry out mutual reactions among a large number of reagents on a flat substrate in immobilizing the probe. Also, when the DNA chip is used for measurement, it is necessary to apply many times a liquid material to a surface of a flat DNA chip in order to carry out reactions with a sample and to wash the DNA chip surface. In order to apply the above treatment to a flat substrate (or DNA chip), the substrate (or the DNA chip) must be dipped in a reactant solution filling a container. Alternatively, a flow path must be formed on the DNA chip surface by using an additional tool such as the channel block noted above, followed by applying the treatment with the liquid. However, the dipping method gives rise to a problem that each kind of the treating liquid must be used in an excessive amount in the immobilizing step and in the sample measuring step. On the other hand, in the method using a flow path, only a limited region of the surface of the DNA chip having a DNA probe immobilized thereon is used, resulting in failure to utilize sufficiently the formed DNA chip. Further, the DNA chip is an open system in which the probe-formed surface is exposed to the outside, giving rise to the defects that the surface tends to be contaminated and that the DNA chip cannot be handled conveniently.
The conventional DNA chip technology gives rise to an additional problem besides the problems given above. Specifically, the conventional DNA chip technology is certainly effective for performing the sequencing of DNA having an unknown base sequence, but is not adapted for the developed pattern analysis of mRNA which will be important in the future. Let us describe the particular problem in the following.
In recent researches on genes, how to utilize the DNA information obtained by the sequencing is more important in view of the post genome than the sequencing itself. For example, the analysis of the mRNA expression pattern is being carried out vigorously in order to make researches on the gene expression profile. The expression of mRNA relating to a certain gene exhibits a different level of expression depending on each organ. Even in the same cell line, the level of expression differs depending on phase factors, particular disease factors, etc. The analysis of the difference in the level of the mRNA expression in an individual, i.e., analysis of the expression pattern, can be applied in various technical fields such as gene diagnosis, gene therapy, development of medicines and agricultural and stock raising industries and, thus, a further progress is expected.
In general, a quantitative analysis of mRNA relating to a target gene is required for the analysis of the expression pattern of mRNA. Employed for the quantitative analysis is a method in which mRNA to be measured or cDNA thereof, which is obtained by a reverse transcription from mRNA, is reacted with a DNA probe having a base sequence complementary thereto so as to hybridize both of them and, thus, to achieve detection. In such a research, a plurality of expression levels of mRNA are measured. Therefore, used are a plurality of DNA probes corresponding to these plural target mRNA molecules. It should also be noted that, in a research on the expression pattern of mRNA, the base sequence of the mRNA or cDNA to be measured is known to some extent, making it possible to use a relatively long DNA probe having about 20 to 60 bases, preferably, about 40 bases, in view of the accuracy and efficiency of the measurement. As a matter of fact, such a relatively long DNA probe is actually used. In order to prepare a probe having such a length by the conventional DNA chip technology, a tremendous labor is required such that it is necessary to prepare 80 to 240 masks and to repeat the base synthesis by lithography and photoreaction 80 to 240 times. Further, in order to improve the measuring accuracy, it is necessary to align the length of the DNA probe. In the case of employing the conventional method, it is substantially impossible to synthesize a DNA probe having an aligned base length directly on a substrate in view of the yield of synthesis in each stage.
On the other hand, about 20,000 to 30,000 kinds of genes of about 100,000 genes present in a single cell are considered to express mRNA. Since a ratio of cell specific genes that are important in the research of the expression pattern is estimated at about several percent (about 1 to 3%), about several hundred to thousand of mRNA or their cDNA are considered to be capable of providing a target object to be measured. According to the conventional DNA chip technology described previously, 48 kinds, i.e., 65,536 kinds, of probes each having a length of 8 bases can be formed on a single substrate. However, since a maximum of about 20,000 to 30,000 kinds of mRNA, actually not larger than {fraction (1/10)} thereof, are considered to be capable of providing the target object, such a tremendous number of kinds of probes need not be formed in practice.
Further, the hybridization condition between a DNA probe and a target object of an mRNA (or its cDNA) segment is not uniform and requires the stringency in the reaction. In addition, it is impossible in view of the dynamic range of the measuring apparatus to process in a single operation a test sample containing a tremendous number of kinds of target mRNA (or cDNA) pieces widely different from each other in concentration to detect the target mRNA. Therefore, even if a tremendous number of kinds of DNA probes are prepared under the same measuring conditions, only a very small proportion of the DNA probes produce a useful result. Such being the situation, a tremendous number of kinds of DNA probes need not be formed on the same substrate.
Further in the conventional DNA chip technology described previously, the synthesis by photoreaction must be repeated for forming the DNA probe, with the result that deterioration of the DNA probe caused by ultraviolet light remains as a problem to be solved. It follows that the conventional DNA chip technology is unsuitable for use in preparation of a DNA probe having a length of 20 bases or more.
Known vessels and methods are also disclosed in several publications. For example, U.S. Pat. No. 5,607,646 discloses a plate capable of pooling a liquid and having a plurality of DNA probes immobilized on a flat bottom. Also, U.S. Pat. No. 5,922,604 discloses a closed vessel, which is circular and has a flat working area. The inflow and outflow of a sample or the like into and out of the working area are performed in the prior art by utilizing the capillary phenomenon. The working area is formed such that the capillary phenomenon is intensified toward the inner region of the working area. Further, a method of detecting DNA by a capillary electrophoresis is disclosed in xe2x80x9cProc. Natl. Acad. Sci. USA, Vol. 91, pp 11348-11352. In this method, there is no immobilized phase of the probe in the fluid passageway. Also, since the detecting apparatus is immobilized in a predetermined position, the detection is performed at the same site. It follows that this apparatus simply permits detecting the size of the molecule separated by the electrophoresis, and the detection is performed only when the separated molecule passes through the detecting point. However, the methods and the vessels disclosed in these publications are incapable of resolving the problems described above.
A first object of the present invention is to carry out the reaction and/or detection of a plurality of target DNA""s at a time promptly under predetermined conditions. And the detection can be performed easily.
A second object of the present invention is to provide a DNA probe apparatus strong against contamination, easy to handle and excellent in efficiency.
A third object of the present invention is to provide a DNA probe apparatus which permits immobilizing the DNA probe without requiring excessive amounts of various processing liquids and also permits enlarging the area that can be utilized.
Further, a fourth object of the present invention is to provide a DNA capillary adapted for analysis of the expression pattern of mRNA.
The first to fourth objects can be achieved by a DNA capillary, comprising a fluid passageway having at least a part thereof defined by a wall capable of transmitting light, a plurality of independent probe regions formed in the inner wall of the fluid passageway, and DNA probes immobilized on the probe regions, respectively, the DNA probes differing from each other depending on the probe regions on which the DNA probes are immobilized.
In an aspect of the present invention, the fluid passageway should desirably be a hollow capillary having the end portion left open. More desirably, the fluid passageway should be a cylindrical capillary. Where the fluid passageway is in the form of a cylinder having the end portion alone left open, the DNA capillary forms a closed system, making it possible to provide a DNA capillary very strong against contamination and easy to handle.
In another aspect of the present invention, a plurality of fluid passageways are arranged in an integral form so as to improve the immobilizing treatment of the DNA probe and the processing capability of the test sample. Particularly, where all of the plural fluid passageways are combined to communicate with each other in the vicinity of at least one end portion of the fluid passageway, the various processing liquids can be collectively introduced into or recovered from the plural fluid passageways through the combined portion.
In another aspect of the present invention, it is desirable to arrange the plural probe regions within a single fluid passageway in the form of annular regions apart from each other along the fluid passageway. The particular arrangement permits improving the efficiency because a plurality of different DNA probes are allowed to perform the detecting function simultaneously by simply passing liquid to be treated, which contains a test sample, or a gas for the drying purpose, through the single fluid passageway only once. Further, in the DNA capillary of the present invention, it is desirable for the different DNA probes to be arranged in annular regions formed over the entire circumferential region of the inner wall of the capillary and positioned apart from each other. The particular construction makes it possible to enlarge the area to be utilized, compared with the conventional DNA chip utilizing a flat plane. As a result, the target substance can be caught efficiently even in the case of using a small amount of a test sample.
In the DNA capillary of the present invention, the fluid passageway can be formed by applying an etching to a glass or silicon substrate. The etching method is useful when a large number of capillaries are prepared by a single processing. In addition, a large number of DNA probes can be immobilized within a large number of capillaries by utilizing a photochemical reaction.
In another aspect of the present invention, it is possible to manufacture a capillary of the present invention from a plastic material by an injection molding. Use of a plastic material permits suppressing the material cost, the processing cost, etc. Also, the method of immobilizing DNA probes employed in the present invention is not limited to a immobilization by photochemical reaction. Specifically, it is also possible to immobilize the DNA probes in the present invention by the conventional spotting method, e.g., a spotting using an ink jet printer.
What should also be noted is that it is desirable in the present invention to use a DNA probe synthesized in advance with a predetermined base sequence and length. In this case, it is possible to provide a DNA probe adapted for the analysis of the expression pattern of mRNA so as to achieve the fourth object of the present invention. To be more specific, since the probe can be immobilized by applying a photochemical reaction only once, it is possible to immobilize stably a DNA probe having at least 20 bases. It follows that it is possible to provide a detecting apparatus adapted for the research on the mRNA expression pattern, though it was impossible to achieve such a detecting apparatus by utilizing the conventional DNA chip technology.
According to another aspect of the present invention, it is possible to immobilize a plurality of kinds of DNA probes to the fluid passageway noted above in a predetermined amount for each kind and independently of each other depending on the kind. It should be noted that the fluid passageway of the DNA capillary according to the present invention has a predetermined constant cross sectional area over at least the processing region. The xe2x80x9cprocessing regionxe2x80x9d represents the region in which a desired processing is applied to the fluid and denotes at least the region between adjacent probe regions. By making constant the cross sectional area of the processing region, it is possible to fluidize the sample to be processed under the same conditions. The fluid passageway in the present invention is tubular. One probe region is arranged circular on the inner wall in a manner to make one complete rotation around the periphery of the fluid contained in the capillary, thereby detecting a desired nucleic acid with a high sensitivity and smoothly.
According to still another aspect of the present invention, it is possible for the cross sectional shape of the fluid passageway to be bent at least partially. To be more specific, it is possible for the cross section of the capillary to be elliptical, circular, arcuate, rectangular, rectangular with one or more roundish corners, or to have a cross sectional shape obtained by combination of these cross sectional shapes. Such shapes are effective for preventing the bubbles from staying in the fluid passageway. Particularly, the shape defined above is effective for preventing the bubble generation and growth under high temperature conditions employed in the DNA reaction, e.g., high temperature equal to the melting point. It is desirable for the cross section of the fluid passageway to be shaped rectangular with roundish corners because the capillary can be manufactured easily and the bubble formation can be prevented.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.